Fundamental studies on medical use of oligonucleotide (for example, antisense oligomer) have recently been actively carried out, and are now entering a stage of concretely searching for potentiality thereof. Actually, there are available many reports of in vitro studies on oligonucleotide: stability against solvent, selection of a target gene sequence to antisense oligomer, membrane permeability in cell lines, life span, nuclease resistance, intercellular distribution and the like.
Along with the progress of these fundamental studies, it has come to be considered very important to select an appropriate carrier substance when using oligonucleotide as a medical drug, and development of such a carrier substance and solution of problems for this purpose are now becoming inevitable tasks. More specifically, for example, important problems for this carrier include exclusion of positive charge of carrier complex which is unfavorable in biological dynamics of administration pathways such as intravenous injection, establishment of an efficient targeting system including cell membrane permeability, and improvement of life span and stability of oligonucleotide drugs and improvement of uptake efficient into cells.
Since oligonucleotide itself is very unstable and susceptible to decomposition under the effect of external conditions, research efforts are actively made to develop a drug delivery carrier for stably and efficiently carrying oligonucleotide to a target organ.
Overview of the process of the past research and development demonstrates that many of the conventional carrier are based on liposome which is a structure having a lipid bilayer configuration similar to a biological membrane. For example, an oligonucleotide drug included in liposome is reported, in which the oligonucleotide is stable in liposome, and at the same time, chemical or cellular specificity of the oligonucleotide is increased by chemical modification from liposome (Alain R. Thierry and Anatoly Dritschils, Nucleic Acid Res., 20, 5691-5698(1992)).
However, fine particle of liposome have a demerit of having a short half-life in blood. To overcome this disadvantage, improvements have recently been made in chemical stability such as oxidation stability, biological stability and colloidal chemical stability, and a new-generation liposome having a long half-life and an immunoliposome (a liposome having an antibody imbedded onto the surface thereof) are being developed, while practical problems have not however as yet been solved.
On the other hand, research efforts have suddenly increased which propose a technique of, by the utilization of anionicity of oligonucleotide, bonding oligonucleotide to a cationic natural protein or a cationic synthetic poly-amino acid, and delivering the resultant ionic complex to or into target cells (Nature, 271, 130-135 (1978), J. Biol. Chem., 262, 4429-4432 (1987), J. Biol. Chem., 263, 14621-14624 (1988), Proc. Natl. Acad. Sci. USA, 87, 3410-3414 (1990)). This technique is based on the fact that oligonucleotide is bonded in terms of charge to a cationic side chain of amino acid such as .epsilon.-amino group of lysine (Lys) residue, for example, and forms a relatively stable complex (Lemaitre, M., et al., Proc. Natl. Acad. Sci. U.S.A., 84, 648-651(1987)). There is however a problem in that formation of these complexes leads to generation of precipitation. In all these studies, therefore, a limit is that complexes are cationic heterogeneous ones.
For example, poly-L-Lys (PLL) is a well-known drug carrier (H. J. P. Ryser and W. C. Shen, Proc. Natl. Acad. Sci. U.S.A., 75, 3867-3879 (1978)). It is known that the complex of PLL and oligonucleotide is biologically very stable, and formation of complex with PLL makes oligonucleotide present stably against nuclease (B. Bayard, et al., Eur. J. Biochem., 151, 319-325(1985)). However, the mixing concentrations in bonding of both PLL and oligonucleotide are limited by the problem of precipitation. This in contrast suggests a possibility that a solution of the complex-precipitation problem increases their mixing concentrations, thus leading to an improved stability of oligonucleotide.
In addition, it is reported that uptake of an oligonucleotide into cell is promoted by forming it to a complex with PLL (Wu, G.Y. and Wu, C.H., J. Biol. Chem., 27, 887-892(1988)). There is available another report showing the fact that partial chemical modification of PLL increases specificity of oligonucleotide to various cells (E. Wanger, et al., Proc. Natl. Acad. Sci. U.S.A., 87, 3410-3414 (1990)). While its action mechanism has not as yet been clarified, a report demonstrates that PLL exerts an effect on cell membranes to assist interporation of oligonucleotide, and another, that the complex formed by covalent bond of PLL and oligonucleotide increases consequently affinity to cell membranes (M. Fechheimer, et al., Proc. Natl. Acad. Sci. U.S.A., 84, 84633-8467 (1987)). The problem of precipitation as described above has not as yet been solved even with these reports.
As another serious problem, many reports point out cytotoxicity of PLL (J. P. Leonetti, et al., Bio-conjugate Chem., 1, 149-153 (1990)) Zhou, et al. compare a complex of poly-D-Lys (PDL), which is a stereoisomer of PLL, with oligonucleotide and that with PLL, and report that, as a result, the complex of PDL and oligonucleotide is biologically more stable than that with PLL, but is more toxic to cells (X. Zhou, et al., Biochem. Biophys. Acta, 1065, 8-14 (1991)). Although it is thus ascertained that the oligonucleotide complex with PLL is less cytotoxic than that with PDL, the problem of cytotoxicity has not as yet been solved. From the point of view of cytotoxicity, a report points out that PLL having a molecular weight of up to 14,000 exhibits no cytotoxicity (J. P. Leonetti, et al., Bio-conjugate Chem., 1, 149-153 (1990)). This complex is not however suitable as a drug carrier because of the problem of metabolism.
Degols, et al. synthesized an antisense oligonucleotide complementary to the mRNA of tat gene of HIV (Human Immunodeficiency Virus)-1 genome, and studied the tat gene inhibiting effect of the oligonucleotide complex with PLL. They thus ascertained that, in vitro, the complex demonstrated an activity more than 100 times as high as that of antisense alone (P. Degols, et al., Antiviral Res., 17, 279-281 (1992)). Simultaneous use of PLL can thus be deemed to have a gene inhibiting effect of the antisense oligonucleotide.
Under the present circumstances, however, the complex of PLL and oligonucleotide involves a serious problem of very easy agglomeration (J. P. Leonetti, et al., GENE, 72, 323-332 (1988)). For this reason, it has been believed difficult to apply such a cationic natural protein and cationic synthetic poly-amino acid to organism as a drug carrier. Under these circumstances, there has been an increasing demand for the development of a novel oligonucleotide carrier to be transported into body, free from precipitation even after formation of a stable complex.